Media 3: Localization of cortical tissue optical changes during seizure activity in vivo with optical coherence tomography

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1812

Media 2: Localization of cortical tissue optical changes during seizure activity in vivo with optical coherence tomography

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1812

Media 6: Localization of cortical tissue optical changes during seizure activity in vivo with optical coherence tomography

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1812

Data_Sheet_1_Cortical cerebrovascular and metabolic perturbations in the 5xFAD mouse model of Alzheimer’s disease.docx

IntroductionThe 5xFAD mouse is a popular model of familial Alzheimer’s disease (AD) that is characterized by early beta-amyloid (Aβ) deposition and cognitive decrements. Despite numerous studies, the 5xFAD mouse has not been comprehensively phenotyped for vascular and metabolic perturbations over its lifespan.MethodsMale and female 5xFAD and wild type (WT) littermates underwent in vivo18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging at 4, 6, and 12 months of age to assess regional glucose metabolism. A separate cohort of mice (4, 8, 12 months) underwent “vessel painting” which labels all cerebral vessels and were analyzed for vascular characteristics such as vessel density, junction density, vessel length, network complexity, number of collaterals, and vessel diameter.ResultsWith increasing age, vessels on the cortical surface in both 5xFAD and WT mice showed increased vessel length, vessel and junction densities. The number of collateral vessels between the middle cerebral artery (MCA) and the anterior and posterior cerebral arteries decreased with age but collateral diameters were significantly increased only in 5xFAD mice. MCA total vessel length and junction density were decreased in 5xFAD mice compared to WT at 4 months. Analysis of 18F-FDG cortical uptake revealed significant differences between WT and 5xFAD mice spanning 4–12 months. Broadly, 5xFAD males had significantly increased 18F-FDG uptake at 12 months compared to WT mice. In most cortical regions, female 5xFAD mice had reduced 18F-FDG uptake compared to WT across their lifespan.DiscussionWhile the 5xFAD mouse exhibits AD-like cognitive deficits as early as 4 months of age that are associated with increasing Aβ deposition, we only found significant differences in cortical vascular features in males, not in females. Interestingly, 5xFAD male and female mice exhibited opposite effects in 18F-FDG uptake. The MCA supplies blood to large portions of the somatosensory cortex and portions of motor and visual cortex and increased vessel length alongside decreased collaterals which coincided with higher metabolic rates in 5xFAD mice. Thus, a potential mismatch between metabolic demand and vascular delivery of nutrients in the face of increasing Aβ deposition could contribute to the progressive cognitive deficits seen in the 5xFAD mouse model.</p

Ceftriaxone is neuroprotective during infection with <i>Toxoplasma</i>.

<p>Treatment of infected mice with ceftriaxone was conducted for one week starting at 5 weeks post-infection and compared to naïve and untreated infected mice. A minimum of 3 mice were used in each group and the experiments were repeated at least once. A) Immunohistochemistry for NeuN on 12μm thick frontal cortex frozen slices (scale bar: 80μm). Inserts: high magnification of NeuN immunohistochemistry (scale bar: 5μm) B) and C) DiI labeling of dendritic spines quantified in naïve, infected and ceftriaxone treated animals (Student’s t-test; infected vs ceftriaxone: p = 0.0004) (scale bars: 9μm).</p

Infection with <i>Toxoplasma</i> disrupts neuronal networks and changes behavior.

<p>A)-D) Naïve (n = 13), 6 week infected (n = 11) and ceftriaxone treated (n = 11) mice were placed in the center of an elevated plus maze and recorded for 5 minutes. B) percentage, C) frequency of time spent in the open arm of the maze and D) velocity and total distance traveled was measured between naive and infected animals. E) EEG raw traces for naïve, 6 week infected and ceftriaxone treated animals (scale bar: 1sec; dots illustrate the peak of one full rhythmic cycle). F) Approximate entropies, Student’s t-test (naïve vs infected, p<0.0001; infected vs ceftriaxone, p = 0.0057; naïve vs ceftriaxone, p = 0.0138) G) EEG power density spectrum for naïve, 4, 5 and 6-week infected and ceftriaxone treated animals. A minimum of 3 animals were used for each group and the experiments repeated twice. Asterisks represented are Bonferonni’s multiple comparison test (infected vs naive) and (ceftriaxone vs infected) H) EEG percent power calculated from power density spectral analysis in G.</p

Ceftriaxone specifically rescues the glutamate transporter GLT-1.

<p>Treatment of infected mice with ceftriaxone was conducted for one week starting at 5 weeks post-infection and compared to naïve and untreated infected mice. Western blot of whole forebrain lysates for A) GLT-1 and B) GS on naïve (n = 3), infected (n = 5) and ceftriaxone treated (GLT-1 n = 5; GS n = 7) animals (Student’s t-test: GLT-1 naive vs. infected: p = 0.0144; GLT-1 infected vs. ceftriaxone: p = 0.0172; GS naïve vs. infected: p = 0.0002; GS infected vs. ceftriaxone: ns). C) Immunohistochemistry for GLT-1 and GFAP on 12μm thick frontal cortex sections (scale bar: 80μm). D) Glutamate and glutamine concentrations in the extracellular space of naïve (n = 4), infected (n = 6) and ceftriaxone (n = 3) treated mice as measured by microdialysis and LCMS (Student’s t-test: naive vs. infected: p = 0.0003; infected vs. ceftriaxone: p = 0.0072).</p

Infection induces chronic astrocytic morphological and molecular changes.

<p>C57Bl/6 mice were infected with <i>Toxoplasma</i> and brains harvested. A) Scanning serial electron microscopy images analyzed for astrocytic endfeet width (highlighted in yellow). Quantification of endfoot width performed over the course of infection (scale bar: 5μm, BV: blood vessel). 6–10 Z stacks containing blood vessels (naïve n = 20; 3 weeks n = 75; 6 weeks n = 134; 12 weeks n = 82) 5–6μm wide were selected and average astrocyte endfoot width was quantified by measuring perivascular astrocyte area and dividing by the blood vessel circumference. Significance compared to naïve *** = p<0.001. The first panel depicts a <i>Toxoplasma</i> cyst inside a neuron (red) within the frontal cortex (unshaded micrographs provided in S1) B) RT-qPCR was performed on whole forebrain RNA with primers for GLT-1, glutamine synthetase (GS) and GLAST over the course of infection and is presented as fold increase over naïve.</p

Glutamate extracellular concentrations increase during infection.

<p>Microdialysis was performed over the course of <i>Toxoplasma</i> infection taking measurements prior to (N) and after infection as indicated (n = 13 biological replicates (3 prior to infection; 2 for each time point thereafter)). A) Hematoxylin and eosin staining of microdialysis probe placement in the frontal cortex. B) Intraperitoneal injections of pentylenetetrazol (PTZ) to determine sensitivity of amino acid (<i>A-V; arrows</i>) detection. C, D) LC-MS analysis on microdialysis samples over the course of infection. One-way ANOVA: Glutamate (p = 0.0003), Arginine (p<0.0001), Proline (p = 0.0168), Serine (p = 0.04), and Tyrosine (p = 0.0149). A Dunnett’s post-test was performed for all timepoints against naïve concentrations and significance shown as asterisks. ‘ND’ indicates samples were below the limits of detection. Amino acids not listed did not change significantly. Essential amino acids are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005643#ppat.1005643.s002" target="_blank">S2 Fig</a>.</p

Ceftriaxone does not alter the immune response to <i>Toxoplasma</i> infection.

<p>Treatment of infected mice with ceftriaxone was conducted for one or three weeks starting at 5 weeks post-infection and compared to naïve and untreated infected mice. A) Parasite burden was quantified by RT-PCR of forebrain DNA extracted at one week post treatment (6 weeks post infection) from infected (n = 4) and ceftriaxone (n = 5) treated animals (Student’s t-test: infected vs. ceftriaxone p = 0.2730) and at three weeks post treatment (8 weeks post infection)(Student’s t-test: infected (n = 5) vs. ceftriaxone (n = 4) p = 0.2902). B) Brain mononuclear cells (BMNC) were extracted following one week of treatment from infected (n = 4) and ceftriaxone (n = 5) treated brains and counted (Student’s t-test: infected vs. ceftriaxone p = 0.0826) and after 3 weeks of ceftriaxone treatment (Student’s t-test: infected (n = 5) vs. ceftriaxone (n = 4) p = 0.7794). C) Hematoxylin and eosin staining was performed on 3 week treated mice and images of the frontal cortex (10X) and blood vessels (25X) within the frontal cortex were taken. D) Flow cytometry of BMNC reveals the lymphocyte (CD45⁺CD11b^-), macrophage (CD45⁺CD11b^hi) and microglial (CD45^intCD11b^int) populations following 3 weeks of treatment with ceftriaxone. E) Quantification of the proportions of BMNC between infected (n = 5) and ceftriaxone (n = 4) treated mice for microglia (p = 0.7804), macrophages (p = 0.5013) and lymphocytes (p = 0.4164). F) Flow cytometry of BMNC for CD4 and CD8 (Student’s t-test: CD4 infected vs. ceftriaxone p = 0.2075; CD8 infected vs. ceftriaxone p = 0.4122). G) Immunohistochemistry for the microglial marker Iba-1 and the astrocytic marker GFAP was performed on 12μm frozen sections from the frontal cortex (scale bar: 80μm); inserts, high magnification images of cell morphology (scale bar: 5μm).</p